Previous work has shown that ablation of nonmuscle myosin heavy chain (NMHC) II-A in mice results in defects in cell adhesion and loss of E-cadherin and beta-catenin localization to cell-cell adhesion sites. Here we study the function of NMHC II-B in mouse embryonic fibroblasts (MEFs), a cell line that normally contains myosin II-A and II-B, but not myosin II-C. We compared a MEF cell line that was derived from a mouse that had been ablated for NMHC II-B with a cell line derived from a wild-type mouse. The myosin II-B ablated MEFs showed a decrease in the content of both beta-1 and beta-3-integrins compared to wild-type cells using immunoblot and FACS analysis. There was no change in alpha-2 and alpha-3-integrins in these cells. Of note, the II-B ablated MEFs showed a decreased attachment to a number of extracellular matrix proteins including type I collagen, fibronectin and laminin compared to wild-type MEFs. The defect in both beta-1-integrin content and cell attachment could be rescued by transfection of II-B ablated MEFs with GFP-NMHC II-B. When siRNA to NMHC II-B was used to lower the content of myosin II-B in wild-type MEFs, there was a reduction in beta-1-integrin as well as a decrease in cell-matrix attachment in the siRNA-treated cells. Interestingly, MEFs that contain a mutant myosin II-B (a single amino acid mutation, Cys in place of Arg 709) that has a decreased actin-activated MgATPase activity and in vitro motility, do not show any abnormality in integrin content or matrix attachment. This implies that myosin motor activity may not be required for beta-integrin expression. Our results suggest that NMHC II-B is involved in the attachment of MEFs to the extracellular matrix by regulating beta-integrin stability.